The hepatic cytochromes P450 (P450s) are endoplasmic-reticulum membrane-anchored enzymes engaged in the breakdown of endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. Exposure to these agents can increase liver P450 content due to increased formation, or reduce it due to inactivation/destruction and/or proteolytic degradation. Such drug-mediated modulation of P450 content can significantly influence clinical drug-drug interactions (DDIs). Thus, clinically relevant DDIs often emerge from altered P450 turnover elicited by drug-mediated P450 stabilization i.e. ethanol (EtOH), as well as enhanced drug-mediated P450 degradation (i.e. grapefruit juice furanocoumarins). We have recently shown that the degradation of P450s such as CYP3A4, the major human liver and intestinal P450, and the EtOH-metabolizing CYP2E1 incurs ubiquitination by gp78 and CHIP E3 ubiquitin (Ub)-ligases and the 26S proteasome (UPD) which leads to their accelerated cellular disposal. Our in vitro studies reveal that such gp78- and CHIP- mediated P450 ubiquitination is considerably enhanced by their multisite protein phosphorylation. The ubiquitinated P450 Lys-residues reside in negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) clusters. This leads us to hypothesize that protein phosphorylation by enhancing the negatively charged character of these clusters enhances P450 molecular recognition by these E3 Ub-ligases and thus would control its UPD. Thus our first specific aim is to test this hypothesis by site-directed mutagenesis of the relevant D/E and S/T residues in each cluster along with a state-of-the-art proteomic approach to determine their relevance to P450 ubiquitination by these E3s. In addition, we have also found that gp78- and CHIP- knockdown via RNA-interference in rat hepatocytes results in increased levels of functionally active hepatic P450s. While this finding is clinically relevant for hepatic drug metabolism and DDIs, we propose that it may also be pathophysiologically significant: In the absence of relevant drug substrates, these P450s through futile oxidative cycling could generate proteotoxic reactive O2-species (ROS), and thus partly contribute to age- dependent oxidative damage and elevated lipid peroxidation observed in the liver and other tissues of CHIP-/- mice. Thus our second specific aim is to explore whether elevated hepatic P450s upon CHIP knockdown contribute to this pathology in CHIP-/- mice. Enhanced CYP2E1-dependent EtOH metabolism in alcoholics is also known to similarly generate ROS, hydroxyethyl radicals (HER) and lipid peroxidation products that damage cellular proteins including those of UPD and autophagic-lysosomal degradation pathways involved in CYP2E1 turnover. Our third specific aim is to explore whether EtOH-impaired CYP2E1 turnover by disrupting the normal intracellular CYP2E1 trafficking results in its increased migration to the outer plasma membrane, whereupon it is recognized by the immune surveillance system, engendering pathogenic autoantibodies clinically associated with alcoholic liver disease, drug-induced hepatitis, and hypersensitivity syndromes.